The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein. OLFM4 is normally expressed in a limited number of tissues, including the prostate, but its biological functions in prostate are largely unknown. In this study, we found that OLFM4 mRNA was reduced or undetectable in prostate cancer tissues and prostate cancer cell lines. To study the effects of OLFM4 on prostate cancer progression both in vitro and in vivo, we transfected PC-3 prostate cancer cells with OLFM4 to establish OLFM4-expressing PC-3 cell clones. The OLFM4-expressing PC-3 cell clones were found to have decreased proliferation and invasiveness compared with vector control- transfected PC-3 cells in in vitro studies. In addition, nude mice injected with OLFM4- expressing PC-3 cells demonstrated reduced tumor growth and bone invasion and metastasis compared with mice injected with vector control-transfected PC-3 cells. Mechanistic studies revealed that OLFM4 may exhibit its effects on cancer cell growth, invasion, and metastasis through cathepsin D, as OLFM4 protein reduced cathepsin D protein levels and attenuated cathepsin D-induced cancer cell proliferation. In addition, overexpression of OLFM4 abrogated stromal cell derived factor-1 (SDF-1)-induced PC-3 cell invasiveness in an in vitro Matrigel invasion assay, partially through blocking SDF- 1-mediated AKT phosphorylation. Coimmunoprecipitation and immunofluorescence staining studies in OLFM4-expressing PC-3 cells demonstrated a direct interaction between OLFM4 and cathepsin D or SDF-1. Taken together, these results suggest that OLFM4 negatively interacts with the oncogenic and metastatic molecules cathepsin D and SDF-1 and inhibits prostate cancer growth and bone metastasis.